miR-197-y and novel-m0012-5p modulate Th cell differentiation via STAT4/STAT6 in flounder (Paralichthys olivaceus)

MicroRNAs (miRNAs) play essential regulatory roles in various cellular processes, including antimicrobial responses and immune regulation. This study aimed to investigate the molecular mechanisms by which miR-197-y and novel-m0012-5p modulate the immune process, particularly T helper (Th) cell differentiation, in flounder (Paralichthys olivaceus). We identified that miR-197-y selectively binds to the 3'UTR of PoSTAT4 to downregulate its expression, while novel-m0012-5p specifically binds to the 3'UTR of PoSTAT6, thereby suppressing the expression of PoSTAT6. High expression levels of both PoSTAT4 and PoSTAT6 were observed in gill tissues, and they were also expressed in CD4⁺ T cells. Further analysis demonstrated that PoSTAT4 regulates Th cell differentiation by activating promoters of PoIFN-γ and PoT-bet, whereas PoSTAT6 enhances the promoter of PoGATA3. In vitro transfection experiments confirmed that miR-197-y and novel-m0012-5p can be efficiently internalized by head kidney leukocytes and inhibit the expression of PoT-bet/PoIFN-γ and PoGATA3, respectively. Collectively, these results indicate that miR-197-y and novel-m0012-5p participate in Th cell differentiation by targeting PoSTAT4 and PoSTAT6, respectively, thereby regulating the promoter activity of T cell differentiation-related gene promoters. These results contribute to a deeper comprehension of microRNA-mediated regulatory processes involved in the adaptive immune response of teleost fish.